The objective of this proposal is to give a better understanding of the determinants of virulence in B. fragilis. The aspect of virulence to be investigated is the mechanism and transfer of antimicrobial resistance. Studies of antimicrobial resistance will be directed at transferable clindamycin-erythromycin, tetracycline, and ampicillin resistance. The separate functions of the clindamycin resistance (Clnr) transfer factor pBFTM10 will be analyzed. The EcoRI fragments of pBFTM10, cloned in E. coli, will be used as probes in epidemiological studies and to study transposition. The mechanism of mobilization of cryptic plasmids and Ampr by pBFTM10 will be investigated. The mechanism of clindamycin resistance encoded by pBFTM10 will be defined. The location of the tetracycline resistance (Tetr) transfer element, whether on the chromosome or a large plasmid, will be studied. The phenomenon of tetracycline induction to increased resistance transfer will be investigated. The importance of surface structures in high level Tetr transfer will be studied. Attempts will be made to transfer the Tetr element into E. coli. The location of the ampicillin resistance determinant and the mechanism of its mobilization by pBFTM10 and the Tetr transfer element will be addressed. The mechanism of cefoxitin inactivation by a clinical isolate of B. fragilis will be studied. The second aspect of the proposal is to develop a method other than conjugation to introduce plasmid and chromosomal DNA into B. fragilis. The transformation protocols developed for aerobic and facultative bacteria will be adapted to the anaerobic environment. To minimize the restriction of transforming DNA, the B. fragilis Clnr transfer factor pBFTM10 will be used. Broad host range plasmids such as RP4, which are immune to restriction systems, will also be transferred to B. fragilis for the study of chromosomal mobilization. An important consideration will be to isolate an efficient transformation recipient cell. It will be sought by obtaining mutants lacking periplasmic and/or extracellular nuclease and capsule. The results of these studies should give insight into the mechanism and transfer of resistance determinants in B. fragilis, an important virulence factor.